This proposal seeks to develop methods for overexpressing, purifying and crystallizing membrane-localized transcription control proteins from Vibrio cholerae, the agent of human cholera disease. ToxR and TcpP are bi-topic membrane proteins with cytoplasmic DNA binding/activation domains. They control expression of important virulence factors by virtue of their ability to activate expression of toxT, the activator of genes encoding cholera toxin and toxin-coregulated pilus. The DNA binding/activation domains of ToxR and TcpP are homologous to the winged Helix-Turn-Helix (w-HTH) family of proteins. The structures of other w-HTH domains has been solved, but the unusual membrane topology of ToxR and TcpP, and significant preliminary data aimed at determining how they recognize DNA and activate toxT transcription, compel an interest in solving their crystal structures when bound to operator DNA. Upon completion, this study will enable us to discriminate between distinct hypotheses for how ToxR and TcpP function. Along with determining the structure of ToxR and TcpP, of interest also is overexpression, purification and crystallization of the structures of membrane effector proteins required for the activity of each: ToxS in the case of ToxR, and TcpH in the case of TcpP. The specific aims of the proposal are as follows: (i.) To purify a 6-His tagged version of full length ToxR for crystallization and structural determination with and without its toxT promoter binding site or its binding site in the ompU promoter (a promoter activated by ToxR independently of TcpP) (ii.) To purify a 6-His tagged version of full length TcpP for crystallization and structural determination with and without its toxT promoter-binding site (iii.) To obtain structural data on co-crystals of ToxR and TcpP on the toxT promoter (to determine whether protein-protein interactions affect DNA binding) (iv.) To purify FLAG epitope- tagged versions of ToxS and TcpH for crystallization and structural determination alone or in a co-crystal with ToxR or TcpP